Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions

Journal name:
Nature Nanotechnology
Volume:
11,
Pages:
941–947
Year published:
DOI:
doi:10.1038/nnano.2016.137
Received
Accepted
Published online

Oxygen-depleted hypoxic regions in the tumour are generally resistant to therapies1. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour2 of magnetotactic bacteria3, Magnetococcus marinus strain MC-1 (ref. 4), can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals5, tend to swim along local magnetic field lines and towards low oxygen concentrations6 based on a two-state aerotactic sensing system2. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in severe combined immunodeficient beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

At a glance

Figures

  1. Assessment of the specificity of the MC-1 antibody in HCT116 colorectal xenografts in SCID beige mice.
    Figure 1: Assessment of the specificity of the MC-1 antibody in HCT116 colorectal xenografts in SCID beige mice.

    a,b, To ascertain the specificity of MC-1 antibody, xenografts were injected with either MC-1 (a) or PBS (b). cf, An unrelated IgG isotype (6.6 µg ml–1) was used (c,d) together with polyclonal rabbit MC-1 antibody (e,f) on two adjacent sections of the same xenograft. In both cases, the reaction was revealed using either FITC (green) or Texas Red (red) secondary antibodies. Specific labelling could be observed only in the MC-1 injected xenografts incubated with MC-1 antibody. Control IgG was unable to identify MC-1. These results confirmed the specificity of the antibodies to detect MC-1 cells in tumours.

  2. MC-1 cells are preferentially located in the hypoxic regions of the xenografts.
    Figure 2: MC-1 cells are preferentially located in the hypoxic regions of the xenografts.

    a,b, To determine the exact location of MC-1 with regard to the local oxygen tension in tissue, a hypoxyprobe-specific antibody labelled with biotin was used, where strands and islands of remaining hypoxic tumour tissue give a positive brownish reaction in the vicinity of necrotic areas, as illustrated in b. c, The MC-1 antibodies specifically detect MC-1, which is visualized by staining with anti-rabbit FITC-labelled secondary antibodies (×10 and ×40). In c, adjacent sections of the same xenografts were incubated with MC-1 antibody and a FITC-conjugated specific secondary antibody to label MC-1, thus confirming the presence of abundant bacteria in areas of lowered oxygen concentration (×10 and ×40 images of MC-1 FITC fluorescently labelled), and high-resolution images were acquired manually using a DP71 digital camera mounted on an Olympus BX61 motorized upright microscope with fluorescence (FITC/Texas Red (TXRED)/4′,6-diamidino-2-phenylindole (DAPI)). d, Representative portion of a paraffin-embedded section reprocessed for transmission electron microscopy (TEM), which identifies the MC-1 bacteria according to their typical ultrastructural features including presence of magnetosomes. The representative pictures were selected from stained sections obtained using ten different tumours isolated from ten tumour-bearing mice collected in three independent experiments.

  3. Penetration of live MC-1 cells with and without magnetic field exposure in HCT116 xenografts following a peritumoral injection.
    Figure 3: Penetration of live MC-1 cells with and without magnetic field exposure in HCT116 xenografts following a peritumoral injection.

    a, Magnetotaxis directional control system used to generate the magnetic field necessary to guide the MC-1 cells towards the xenograft. b, MC-1 peritumoral injection into HCT116 tumour xenograft in mice and representation of the applied directional magnetic field used in this study to direct the bacteria towards the xenograft. The directional magnetic field B was aligned towards the centre of the tumoral volume. c,d, MC-1 average distribution in transverse tumour (n = 10) sections visualized by staining with anti-rabbit FITC-labelled secondary antibodies at 2, 4, 6 and 8 mm from the peritumoral injection site for group I (c) and group II (d). The results show not only a significant increase in the targeting ratios of magnetically guided MC-1 cells to non-magnetically guided MC-1 cells, but also a good distribution in the tumoral volume and more specifically in the tumour hypoxic regions. The higher populations of MC-1 cells in group I contribute in targeting all hypoxic regions in the tumour. e, Standard deviation and average number of MC-1 cells in the transverse tumour sections in groups I and II. Statistical analysis was performed using unpaired Student's t-test. Significant difference was considered for **P < 0.01. The results clearly show a significant increase in MC-1 cells for group I in the tumoral volume where magnetotactic directional guidance was used prior to removing the magnetic field to enable aerotactic displacements of the MC-1 cells towards the hypoxic areas once inside the xenograft. Without magnetotactic directional guidance, only a smaller proportion of MC-1 cells, which randomly swam in the direction of the xenograft, could potentially be influenced by oxygen gradients in the tumoral tissues. f, Summary of the total average numbers of injected and detected MC-1 cells for all tumours in groups I and II. Data are shown as mean + s.d. (n = 10 for cf). Transverse tumour sections were scanned at ×40 magnification and the numbers of bacteria in the tumours were estimated using image-processing techniques (see Methods, ‘MC-1 count’). The results show that a significant number of the peritumorally injected MC-1 cells being magnetically guided using a relatively simple static directional magnetic field were able to penetrate the xenograft, unlike the case for bacteria that were not guided into the tumour. These results probably represent a simplified case scenario, as many other more sophisticated modulated directional magnetic fields could be investigated to increase further the targeting ratios achieved in this study.

  4. Superior penetration of MC-1 cells over passive diffusion in HCT116 xenografts demonstrated by two methods.
    Figure 4: Superior penetration of MC-1 cells over passive diffusion in HCT116 xenografts demonstrated by two methods.

    ad, Peritumoral injection of a mixture of MC-1 cells and similar sized polymer fluorescent microspheres. e, Peritumoral injection of a mixture of dead and live MC-1 cells. For both methods, injection was followed by 30 min of magnetic guidance directed towards the centre of the tumoral volume. In a, ×20 images are presented of MC-1 Texas Red fluorescent and FITC fluorescent microspheres at various tumoral depths in a longitudinal tumour section, showing superior penetration of MC-1 cells inside the tumour. The small wide strip marks the site where the tumour slice was cut into two parts: one larger and one smaller rectangle. The quantities of fluorescent microspheres and live MC-1 cells at various tumoral depths (b) from the experiment in a depict the superior penetration of live MC-1 cells well past the diffusion limits of the fluorescent microspheres. In c, ×20 images are presented of MC-1 Texas Red fluorescent and FITC fluorescent beads 1, 3 and 6 mm inside the HCT116 xenograft, again showing the much higher penetration depths of the live MC-1 cells. Co-localization of MC-1 bead complexes at a tumoral depth of 4 mm (d) suggest the capability of MC-1 cells to transport large therapeutic payloads deep into tumoral tissues. Dead MC-1 cells are unable to move in the magnetic field and are located in the far left section, whereas live MC-1 cells, which typically migrate along the magnetic field, are located in the right section, confirming that they indeed move along the direction of the magnetic field. In e, ×20 images are shown of live and dead MC-1 cells at different tumoral depths, showing the superior penetration of live cells into the tumours.

  5. Targeting ratios of MC-1–LP in HCT116 xenografts.
    Figure 5: Targeting ratios of MC-1–LP in HCT116 xenografts.

    a, Scanning electron microscopy images of unloaded MC-1 (left) and MC-1–LP (right) with ∼70 SN-38 loaded liposomes (diameter, ∼170 nm) attached to the surface of each cell. b, Estimated mean ratios of MC-1–LP found in the tumour after targeting with a directional magnetic field. The total numbers of bacteria in the tumours were estimated by a direct count of the MC-1 cells obtained from homogenization of the tumours or image-processing techniques of transverse tumour sections. The data represent mean ± s.d. (n = 10 tumours). The numbers of bacteria were calculated using ten different tumours isolated from ten tumour-bearing mice collected in three independent experiments. Five tumours were homogenized to obtain the exact count and the other five were cut into numerous sections, stained and counted. The preliminary results already show a mean targeting ratio of 55%, similar to the targeting results achieved with unloaded MC-1 cells under the same experimental and physiological conditions. These results suggest that the MC-1 can be loaded without significantly affecting the targeting ratios in tumours. c, Transverse tumour sections of MC-1–LP after targeting. Images of each section were acquired using a fluorescence optical microscope equipped with a ×40 magnification objective lens. The images show a good distribution of the loaded MC-1 cells throughout the tumour. d, Examples of MC-1–LP distributions inside four different tumours. The differences in the locations of the hypoxic regions among the various xenografts lead to variations in the distributions of the loaded MC-1 cells between the targeted xenografts.

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Author information

  1. These authors contributed equally to this work

    • Ouajdi Felfoul,
    • Mahmood Mohammadi,
    • Samira Taherkhani &
    • Danuta Radzioch

Affiliations

  1. NanoRobotics Laboratory, Department of Computer and Software Engineering, Institute of Biomedical Engingeering, Polytechnique Montréal, Montréal H3T 1J4, Canada

    • Ouajdi Felfoul,
    • Mahmood Mohammadi,
    • Samira Taherkhani,
    • Dominic de Lanauze,
    • Dumitru Loghin,
    • Neila Kaou,
    • Michael Atkin &
    • Sylvain Martel
  2. Department of Biomedical Engineering, McGill University, Montréal H3A 2B4, Canada

    • Samira Taherkhani,
    • Sherief Essa &
    • Maryam Tabrizian
  3. McGill University Health Centre, Montréal H4A 3J1, Canada

    • Yong Zhong Xu,
    • Sylwia Jancik,
    • Daniel Houle &
    • Danuta Radzioch
  4. Department of Chemistry, University of Montréal (UdM), Montréal H3C 3J7, Canada

    • Sherief Essa &
    • Michel Lafleur
  5. Department of Pathology and Cell Biology, Institute for Research in Immunology and Cancer (IRIC), University of Montréal, Montréal H3T 1J4, Canada

    • Louis Gaboury
  6. Faculty of Dentistry, McGill University, Montréal H3A 1G1, Canada

    • Maryam Tabrizian
  7. Department of Oncology, Segal Cancer Centre, Jewish General Hospital, McGill University, Montréal H3T 1E2, Canada

    • Té Vuong &
    • Gerald Batist
  8. Departments of Biochemistry, Medicine and Oncology, Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montréal H3A 1A3, Canada

    • Nicole Beauchemin

Contributions

S.M. acted as principal investigator, wrote the paper with assistance from O.F., M.M., D.R., S.T. and N.B. and developed the general principles and methods. G.B. and T.V. provided clinical insights. O.F., M.M., S.T., Y.Z.X., D.d.-L., D.L. and D.H. performed the experiments with tumour-bearing animals. S.J., Y.Z.X. and D.H. carried out the IV injections, processed all blood and tissues for analysis and analysed the samples. L.G. performed immunohistochemistry, immunofluorescence and histopathological evaluations. N.K. and M.M., assisted by M.A., acted as project managers. M.M. and O.F. performed the experiments for the preliminary in vivo proofs of concept. O.F. designed the experimental platform. D.d.-L. tested the magnetotactic control process, which was executed by D.L. M.L. and S.E. synthesized the liposomes. M.T. and S.T. attached the liposomes to the MC-1 cells. M.M. cultivated and prepared the bacteria for injection. M.M., D.R., G.B., L.G., N.B. and S.M. made revisions to the manuscript and figures. Y.Z.X. and D.H. coordinated the implantations of tumour xenografts. D.L. developed and calibrated the MC-1 counting software.

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The authors declare no competing financial interests.

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